Fiber type composition of unoperated rat soleus and extensor digitorum longus muscles after unilateral isotransplantation of a foreign muscle in long-term experiments

We examined the effects of the unilateral heterochronous isotransplantation on the fiber type composition and myosin heavy chain (MyHC) isoform content of unoperated slow soleus and fast extensor digitorum longus muscles of female inbred Lewis strain rats. Comparison was made between "control" unoperated muscles of experimental rats (after intramuscular transplantation surgery) with the corresponding muscles of completely naive (unoperated) rats of three age groups (5-, 8- and 14-month-old). This was done in order to ascertain whether these muscles can be used as reliable controls to the transplanted and host muscles for our ongoing grafting experiments. The fiber type composition was determined by assessing the histochemical reaction for myofibrillar adenosine triphosphatase, the MyHC isoform content was determined immunocytochemically using monoclonal antibodies specific to different MyHC isoforms and by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Our experiments show that the heterochronous intramuscular isotransplantation procedure had no significant effect on the fiber type composition and MyHC isoform content of the "control" unoperated muscles of the experimental rats when compared to the corresponding muscles of the naive animals. Furthermore, the duration and type of isotransplantation did not also lead to differences among corresponding "control" muscles of experimental animals. We conclude that the unoperated muscles of the experimental rats can be used as controls in our current transplantation project dealing with long-term grafting experiments.     

Alterations of tension-dependent ATP utilization in a transgenic rat model of hypertrophic cardiomyopathy

Although it is established that familial hypertrophic cardiomyopathy (FHC) is caused by mutations in several sarcomeric proteins, including cardiac troponin T (TnT), its pathogenesis is still not completely understood. Previously, we established a transgenic rat model of FHC expressing a human TnT molecule with a truncation mutation (DEL-TnT). This study investigated whether contractile dysfunction and electrical vulnerability observed in DEL-TnT rats might be due to alterations of intracellular Ca(2+) homeostasis, myofibrillar Ca(2+) sensitivity, and/or myofibrillar ATP utilization. Simultaneous measurements of the force of contraction and intracellular Ca(2+) transients were performed in right ventricular trabeculae of DEL-TnT hearts at 0.25 and 1.0 Hz. Rats expressing wild-type human TnT as well as nontransgenic rats served as controls. In addition, calcium-dependent ATPase activity and tension development were investigated in skinned cardiac muscle fibers. Force of contraction was significantly decreased in DEL-TnT compared with nontransgenic rats and TnT. Time parameters of Ca(2+) transients were unchanged at 0.25 Hz but prolonged at 1.0 Hz in DEL-TnT. The amplitude of the fura-2 transient was similar in all groups investigated, whereas diastolic and systolic fura-2 ratios were found elevated in rats expressing nontruncated human troponin T. In DEL-TnT rats, myofibrillar Ca(2+)-dependent tension development as well as Ca(2+) sensitivity of tension were significantly decreased, whereas tension-dependent ATP consumption ("tension cost") was markedly increased. Thus, a C-terminal truncation of the cardiac TnT molecule impairs the force-generating capacity of the cycling cross-bridges resulting in increased tension-dependent ATP utilization. Taken together, our data support the hypothesis of energy compromise as a contributing factor in the pathogenesis of FHC.     

Phosphodiesterase type 5 inhibitor sildenafil citrate does not potentiate the vasodilative properties of nebivolol in rat aorta

BACKGROUND: Sildenafil citrate (SIL) is contraindicated in patients with coronary heart disease who are treated with nitric oxide (NO) donators such as organic nitrates, as it potentiates NO-mediated vasodilation. The present study investigated whether SIL also affects the vasodilatory effects of nebivolol (NEB), a selective beta1-adrenoceptor blocker with an additional, endothelium-dependent NO-liberating property, in comparison to the combination SIL/glycerol trinitrate (GTN). METHODS AND RESULTS: Experiments were performed in isolated vessel rings of rat aorta (Wistar rats, 8-12 weeks), which had been pre-contracted with phenylephrine (10(-5) M). Isometric tension was measured by a force transducer, and cumulative concentration-response curves were obtained for each drug. The rank order of vasodilatory potency as measured by the concentration needed to achieve 50% relaxation (EC50) was GTN (0.08 microM) > SIL (1.25 microM) > or = NEB (3.5 microM). In the presence of both therapeutic (1 nM) and high (1 microM) concentrations of SIL, vasodilation of GTN was potentiated as indicated by a significant increase in vasodilatory potency (EC50 GTN + low SIL: 0.019 microM, EC50 GTN + high SIL: 0.002 microM; both P < 0.01 vs. GTN). In contrast, SIL did not potentiate the vasodilatory effect of NEB (EC50 NEB + low SIL: 5.01 microM, EC50 NEB + high SIL: 3.2 microM; n.s. vs. NEB). CONCLUSIONS: These data demonstrate that SIL does not potentiate NEB-induced vasodilation in vitro. These findings indicate that the interaction between SIL and NO-donators/organic nitrates does not apply to the NO-liberating properties of NEB. Our findings suggest that SIL may safely be used in hypertensive patients treated with NEB.     

Whole-genome QTL analysis for MAGIC

Key message

An efficient whole genome method of QTL analysis is presented for Multi-parent advanced generation integrated crosses.

Abstract

Multi-parent advanced generation inter-cross (MAGIC) populations have been developed for mice and several plant species and are useful for the genetic dissection of complex traits. The analysis of quantitative trait loci (QTL) in these populations presents some additional challenges compared with traditional mapping approaches. In particular, pedigree and marker information need to be integrated and founder genetic data needs to be incorporated into the analysis. Here, we present a method for QTL analysis that utilizes the probability of inheriting founder alleles across the whole genome simultaneously, either for intervals or markers. The probabilities can be found using three-point or Hidden Markov Model (HMM) methods. This whole-genome approach is evaluated in a simulation study and it is shown to be a powerful method of analysis. The HMM probabilities lead to low rates of false positives and low bias of estimated QTL effect sizes. An implementation of the approach is available as an R package. In addition, we illustrate the approach using a bread wheat MAGIC population.     

Effect of solar particle event radiation on gastrointestinal tract bacterial translocation and immune activation

Space flight conditions within the protection of Earth's gravitational field have been shown to alter immune responses, which could lead to potentially detrimental pathology. An additional risk of extended space travel outside the Earth's gravitational field is the effect of solar particle event (SPE) radiation exposure on the immune system. Organisms that could lead to infection include endogenous, latent viruses, colonizing pathogenics, and commensals, as well as exogenous microbes present in the spacecraft or other astronauts. In this report, the effect of SPE-like radiation on containment of commensal bacteria and the innate immune response induced by its breakdown was investigated at the radiation energies, doses and dose rates expected during an extravehicular excursion outside the Earth's gravitational field. A transient increase in serum lipopolysaccharide was observed 1 day after irradiation and was accompanied by an increase in acute-phase reactants and circulating proinflammatory cytokines, indicating immune activation. Baseline levels were reestablished by 5 days postirradiation. These findings suggest that astronauts exposed to SPE radiation could have impaired containment of colonizing bacteria and associated immune activation.     

Abacavir Induced T Cell Reactivity from Drug Na?ve Individuals Shares Features of Allo-Immune Responses

Abacavir hypersensitivity is a severe hypersensitivity reaction which occurs exclusively in carriers of the HLA-B*57∶01 allele. In vitro culture of PBMC with abacavir results in the outgrowth of abacavir-reacting CD8⁺ T cells, which release IFNγ and are cytotoxic. How this immune response is induced and what is recognized by these T cells is still a matter of debate. We analyzed the conditions required to develop an abacavir-dependent T cell response in vitro. The abacavir reactivity was independent of co-stimulatory signals, as neither DC maturation nor release of inflammatory cytokines were observed upon abacavir exposure. Abacavir induced T cells arose in the absence of professional APC and stemmed from naïve and memory compartments. These features are reminiscent of allo-reactivity. Screening for allo-reactivity revealed that about 5% of generated T cell clones (n = 136) from three donors were allo-reactive exclusively to the related HLA-B*58∶01. The addition of peptides which can bind to the HLA-B*57∶01-abacavir complex and to HLA-B*58∶01 during the induction phase increased the proportion of HLA-B*58∶01 allo-reactive T cell clones from 5% to 42%. In conclusion, abacavir can alter the HLA-B*57∶01-peptide complex in a way that mimics an allo-allele (‘altered self-allele’) and create the potential for robust T cell responses.     

Detection of Puumala Hantavirus Antigen in Human Intestine during Acute Hantavirus Infection

Background

Puumala virus (PUUV) is the most important hantavirus species in Central Europe. Nephropathia epidemica (NE), caused by PUUV, is characterized by acute renal injury (AKI) with thrombocytopenia and frequently gastrointestinal symptoms.

Methods

456 patients with serologically and clinically confirmed NE were investigated at time of follow-up in a single clinic. The course of the NE was investigated using medical reports. We identified patients who had endoscopy with intestinal biopsy during acute phase of NE. Histopathological, immunohistochemical and molecular analyses of the biopsies were performed.

Results

Thirteen patients underwent colonoscopy or gastroscopy for abdominal pain, diarrhea, nausea and vomiting during acute phase of NE. Immunohistochemistry (IHC) revealed PUUV nucleocapsid antigen in 11 biopsies from 8 patients; 14 biopsies from 5 patients were negative for PUUV nucleocapsid antigen. IHC localized PUUV nucleocapsid antigen in endothelial cells of capillaries or larger vessels in the lamina propria. Rate of AKI was not higher and severity of AKI was not different in the PUUV-positive compared to the PUUV-negative group. All IHC positive biopsies were positive for PUUV RNA using RT-PCR. Phylogenetic reconstruction revealed clustering of all PUUV strains from this study with viruses previously detected from the South-West of Germany. Long-term outcome was favorable in both groups.

Conclusions

In patients with NE, PUUV nucleocapsid antigen and PUUV RNA was detected frequently in the intestine. This finding could explain frequent GI-symptoms in NE patients, thus demonstration of a more generalized PUUV infection. The RT-PCR was an effective and sensitive method to detect PUUV RNA in FFPE tissues. Therefore, it can be used as a diagnostic and phylogenetic approach also for archival materials. AKI was not more often present in patients with PUUV-positive IHC. This last finding should be investigated in larger numbers of patients with PUUV infection.     

IL-23 p19 Knockout Mice Exhibit Minimal Defects in Responses to Primary and Secondary Infection with Francisella tularensis LVS

Our laboratory’s investigations into mechanisms of protective immunity against Francisella tularensis Live Vaccine Strain (LVS) have uncovered mediators important in host defense against primary infection, as well as those correlated with successful vaccination. One such potential correlate was IL-12p40, a pleiotropic cytokine that promotes Th1 T cell function as part of IL-12p70. LVS-infected IL-12p40 deficient knockout (KO) mice maintain a chronic infection, but IL-12p35 KO mice clear LVS infection; thus the role that IL-12p40 plays in immunity to LVS is independent of the IL-12p70 heterodimer. IL-12p40 can also partner with IL-23p19 to create the heterodimeric cytokine IL-23. Here, we directly tested the role of IL-23 in LVS resistance, and found IL-23 to be largely dispensable for immunity to LVS following intradermal or intranasal infection. IL-23p19 KO splenocytes were fully competent in controlling intramacrophage LVS replication in an in vitro overlay assay. Further, antibody responses in IL-23p19 KO mice were similar to those of normal wild type mice after LVS infection. IL-23p19 KO mice or normal wild type mice that survived primary LVS infection survived maximal doses of LVS secondary challenge. Thus p40 has a novel role in clearance of LVS infection that is unrelated to either IL-12 or IL-23.     

Cytosolic RNA:DNA hybrids activate the cGAS–STING axis

Intracellular recognition of non‐self and also self‐nucleic acids can result in the initiation of potent pro‐inflammatory and antiviral cytokine responses. Most recently, cGAS was shown to be critical for the recognition of cytoplasmic dsDNA. Binding of dsDNA to cGAS results in the synthesis of cGAMP(2′–5′), which then binds to the endoplasmic reticulum resident protein STING. This initiates a signaling cascade that triggers the induction of an antiviral immune response. While most studies on intracellular nucleic acids have focused on dsRNA or dsDNA, it has remained unexplored whether cytosolic RNA:DNA hybrids are also sensed by the innate immune system. Studying synthetic RNA:DNA hybrids, we indeed observed a strong type I interferon response upon cytosolic delivery of this class of molecule. Studies in THP‐1 knockout cells revealed that the recognition of RNA:DNA hybrids is completely attributable to the cGAS–STING pathway. Moreover, in vitro studies showed that recombinant cGAS produced cGAMP upon RNA:DNA hybrid recognition. Altogether, our results introduce RNA:DNA hybrids as a novel class of intracellular PAMP molecules and describe an alternative cGAS ligand next to dsDNA.